Anti-cancer compounds

ABSTRACT

The present invention relates to anti-cancer compounds, methods for their discovery, and their therapeutic use. In particular, the present invention provides analogs of the known anti-cancer compound amonafide, and structurally and functionally related compounds, and methods of using such compounds as therapeutic agents to treat a number of conditions associated with hyperproliferation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to pending U.S. Provisional PatentApplication No. 61/020,626 filed Jan. 11, 2008, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to anti-cancer compounds, methods fortheir discovery, and their therapeutic use. In particular, the presentinvention provides analogs of the known anti-cancer compound amonafide,and structurally and functionally related compounds, and methods ofusing such compounds as therapeutic agents to treat a number ofconditions associated with hyperproliferation.

BACKGROUND

Cancer is a group of diseases in which cells are aggressive (grow anddivide without respect to normal limits), invasive (invade and destroyadjacent tissues), and/or metastatic (spread to other locations in thebody). These three malignant properties of cancers differentiate themfrom benign tumors, which are self-limited in their growth and do notinvade or metastasize (although some benign tumor types are capable ofbecoming malignant). Cancer may affect people at all ages, even fetuses,but risk for the more common varieties tends to increase with age.Cancer causes about 13% of all deaths. Improved methods for treatingcancer are needed.

SUMMARY OF THE INVENTION

The present invention relates to anti-cancer compounds, methods fortheir discovery, and their therapeutic use. In particular, the presentinvention provides analogs of the known anti-cancer compound amonafide,and structurally and functionally related compounds, and methods ofusing such compounds as therapeutic agents to treat a number ofconditions associated with hyperproliferation.

The present invention relates to metabolically stable analogs of a knownanit-cancer drug, amonafide. In order to eliminate the metabolicinstability of amonafide while retaining the anti-cancer properties ninederivatives that are structurally similar to amonafide that should notbe acetylated were synthesized. Eight derivatives have aryl amines atthe 6-position (vs. 5-position of amonafide) and one derivativecompletely lacks the aryl amine. These derivatives can evade metabolismby NAT2, which will hence likely improve the clinical management ofpatients compared to those treated with amonafide. The analogs reportedhere may serve as a clinical replacement for amonafide or may be used anovel metabolically stable anti-cancer drugs.

Accordingly, in certain embodiments, the present invention providescompositions comprising a compound described by the following formula:

including salts, esters, and prodrugs thereof; and including both R andS enantiomeric forms and racemic mixtures thereof; wherein X is presentor absent, and if present is

wherein R is H, ethyl, or R1, and wherein R1 is H, ethyl,

The present invention is not limited to a particular type of compound.In some embodiments, the compound is one of the following compounds:

In certain embodiments, the present invention provides pharmaceuticalpreparations comprising one or more of the compounds and apharmaceutically acceptable carrier. In some embodiments, the presentinvention provides methods for treating a hyperproliferative disordercomprising administering an effective amount of the pharmaceuticalpreparation to a subject in need thereof. The present invention is notlimited to a particular type of hyperproliferative disorder. In someembodiments, the hyperproliferative disorder is a cancer (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, bladder cancer, leukemia cancer, prostate cancer, renalcancer, uterine cancer, ovarian cancer, breast cancer, colon cancer,cervical cancer, and lung cancer). In some embodiments, the methodscomprise co-administering to the subject an anti-cancer agent (e.g.,Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin;Adriamycin; Aldesleukin; Alitretinoin; Allopurinol Sodium; Altretamine;Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine;Anastrozole; Annonaceous Acetogenins; Anthramycin; Asimicin;Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat;Benzodepa; Bexarotene; Bicalutamide; Bisantrene Hydrochloride; BisnafideDimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine;Bullatacin; Busulfan; Cabergoline; Cactinomycin; Calusterone;Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Celecoxib; Chlorambucil;Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; DACA(N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin;Daunorubicin Hydrochloride; Daunomycin; Decitabine; Denileukin Diftitox;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized OilI 131; Etoposide; Etoposide Phosphate; Etoprine; FadrozoleHydrochloride; Fazarabine; Fenretinide; Floxuridine; FludarabinePhosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; FostriecinSodium; FK-317; FK-973; FR-66979; FR-900482; Gemcitabine; GeimcitabineHydrochloride; Gemtuzumab Ozogamicin; Gold Au 198; Goserelin Acetate;Guanacone; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-1a; Interferon Gamma-1b; Iproplatin;Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; LeuprolideAcetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine;Losoxantrone Hydrochloride; Masoprocol; Maytansine; MechlorethamineHydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Methoxsalen; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;Mitogillin; Mitomalcin; Mitomycin; Mytomycin C; Mitosper; Mitotane;Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;Oprelvekin; Ormaplatin; Oxisuran; Paclitaxel; Pamidronate Disodium;Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rituximab; Rogletimide; Rolliniastatin;Safingol; Safingol Hydrochloride; Samarium/Lexidronam; Semustine;Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Squamocin; Squamotacin;Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur;Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine;Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene Citrate;Trastuzumab; Trestolone Acetate; Triciribine Phosphate; Trimetrexate;Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; UracilMustard; Uredepa; Valrubicin; Vapreotide; Verteporfin; Vinblastine;Vinblastine Sulfate; Vincristine; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride; 2-Chlorodeoxyadenosine; 2′-Deoxyformycin;9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid;2-chloro-2′-arabino-fluoro-2′-deoxyadenosine;2-chloro-2′-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R;CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlorethamine);cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan;N-methyl-N-nitrosourea (MNU); N,N′-Bis(2-chloroethyl)-N-nitrosourea(BCNU); N-(2-chloroethyl)-N′-cyclohex-yl-N-nitrosourea (CCNU);N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU);N-(2-chloroethyl)-N′-(diethyl)ethylphosphonate-N-nit-rosourea(fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide;temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin;Carboplatin; Ormaplatin; Oxaliplatin; C1-973; DWA 2114R; JM216; JM335;Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine;6-Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide; 9-aminocamptothecin; Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin;darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);amsacrine; pyrazoloacridine; all-trans retinol;14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-Hydroxyphenyl)retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid;fludarabine (2-F-ara-AMP); 2-chlorodeoxyadenosine (2-Cda),Antiproliferative agents, Piritrexim Isothionate, Antiprostatichypertrophy agents, Sitogluside, Benign prostatic hyperplasia therapyagents, Tamsulosin Hydrochloride, Prostate growth inhibitor agents,Pentomone, and Radioactive agents, Fibrinogen 1 125; Fludeoxyglucose F18; Fluorodopa F 18; Insulin I 125; Insulin I 131; Iobenguane I 123;Iodipamide Sodium I 131; Iodoantipyrine I 131; Iodocholesterol I 131;Iodohippurate Sodium I 123; Iodohippurate Sodium I 125; IodohippurateSodium I 131; Iodopyracet I 125; Iodopyracet I 131; IofetamineHydrochloride I 123; Iomethin I 125; Iomethin I 131; Iothalamate SodiumI 125; Iothalamate Sodium I 131; Iotyrosine I 131; Liothyronine I 125;Liothyronine I 131; Merisoprol Acetate Hg 197; Merisoprol Acetate Hg203; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Tc 99mAntimony Trisulfide Colloid; Technetium Tc 99m Bicisate; Technetium Tc99m Disofenin; Technetium Tc 99m Etidronate; Technetium Tc 99mExametazime; Technetium Tc 99m Furifosmin; Technetium Tc 99m Gluceptate;Technetium Tc 99m Lidofenin; Technetium Tc 99m Mebrofenin; Technetium Tc99m Medronate; Technetium Tc 99m Medronate Disodium; Technetium Tc 99mMertiatide; Technetium Tc 99m Oxidronate; Technetium Tc 99m Pentetate;Technetium Tc 99m Pentetate Calcium Trisodium; Technetium Tc 99mSestamibi; Technetium Tc 99m Siboroxime; Technetium Tc 99m Succimer;Technetium Tc 99m Sulfur Colloid; Technetium Tc 99m Teboroxime;Technetium Tc 99m Tetrofosmin; Technetium Tc 99m Tiatide; Thyroxine I125; Thyroxine I 131; Tolpovidone I 131; Triolein I 125; and Triolein I131).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows synthesis of amonafide derivatives. (A) CYP1A2 isresponsible for the inactivation of amonafide and NAT2 is responsiblefor the production of the toxic N-acetyl species. (B) Synthesis schemefor A1-A9. (C) In vitro NAT2 acetylation assay demonstrates 6-positionamino derivatives cannot be acetylated. Recombinant human NAT2 enzymeand mass spectrometry were used to determine if the novel derivativescan be metabolized by this enzyme. Amonafide is a positive control, A1is an unknown as it has a 6-postion free aryl amine, and A2 is arepresentative negative control as it does not have a free aryl amineand therefore cannot be acetylated by NAT2, which requires a substratefree amine for activity.

FIG. 2 shows potencies and selectivity indicies of amonafide andderivatives. (A) Growth inhibition potencies of amonafide and A1-A9against HeLa cells and PBMCs as determined by the MTS proliferationassay. 6-position amino composition or presence is minimally importantfor potencies in cultured cells. (B) Selectivity indices for the samecompounds. The selectivity was calculated according to the formula onthe y-axis so that a positive value corresponds to selective growthinhibition of HeLa cells over PBMCs. The composition of the 6-positionamino group does affect the selectivity of these compounds.

FIG. 3 shows subcellular localization of amonafide (Amn) and A1-A8.These compounds fluoresce in the green channel; therefore, the signal inthe first row represents the relative subcellular localization of eachcompound (Cmpd). The middle row is the DAPI staining to mark the nucleusand the bottom row is Texas Red-Phalloidin (TR-Ph) staining to mark theboundaries of the cytoplasm. The last column is a representative pictureof vehicle treated cells (Veh) to show that the signals observed intreated cells are not due to auto-fluorescence of endogenous cellularcomponents. Scale bars=10 μm. The 6-position amino group is shown beloweach derivative. Differential sub-cellular localization was observedwith the more hydrophobic derivatives localizing heavily to cytoplasmicpuncta while the others preferentially localize to the nucleus. A9 wasincluded in this figure as it lacks the aryl amine and hence does notfluoresce.

FIG. 4 shows DNA intercalation assay data. pBR322 DNA was incubated withvarious concentrations of each derivative or vehicle (1% DMSO) andtoposiomerasel (or water) was added to each reaction. The topoI enzymecannot relax DNA that is unwound by an intercalating agent. Therefore,compounds that intercalate DNA shift the equilibrium of the reactiontowards form I pBR322. A1 and the hydrophobic derivatives (A5-A8)intercalated DNA to a lesser extent than amonafide and the otherderivatives.

FIG. 5 shows in vivo topoisomeraseII inhibition assays. (A) Top panel:Example of fractionation and spectrometry to isolate genomic DNA and thecorrelating slot blot for detection of topoII-DNA crosslinks using apositive (etoposide) and a negative (vehicle −1% DMSO) control. Bottompanel: Quantification of topoII-DNA complexes in HeLa cells treated with1% DMSO (Veh) or [IC_(99%)] of mitoxantrone (Mito −1.01 μM), etoposide(Etop −9.62 μM), camptothecin (Cmpt −280 nM), amonafide (Amn), andamonafide derivatives (A1-A9). Error bars=+SD. Amonafide and A1-A9 donot stabilized topoII-DNA cleavable complexes like other topoIIinhibitors. (B) Quantification of DNA damage response in HeLa cellstreated with the [IC_(99%)] by western blotting for phospho-Thr68 Chk2and total Chk2 protein as a surrogate marker of in vivo topoIIinhibition. The ratio of activated Chk2 was determined by dividing theintensity of the p-Chk2 bands by the intensity of the Chk2 bands (errorbars=SD). Differential DNA damage response was observed among thederivatives. Data from (A) and (B) experiments were normalized to theresponse of mitoxantrone.

FIG. 6 shows the effect of amonafide and A1-A9 on malignant behavior.(A) Transwell invasion assay quantification to measure theanti-metastatic ability of the derivatives. Data were normalized to %invading cells as compared to the vehicle (1% DMSO) control (errorbars=SD). (B) Top panel: HeLa cells treated with 1% DMSO (Veh) or 8.26μM amonafide (Amn) for 20 hr and then immunofluorescently stained withantibodies to PTB (a component of the PNC). PTB localizes to thenucleoplasm (excluding nucleoli) and is highly concentrated in the PNCif present. Exemplary PNCs are marked with arrow heads. Scale bar=10 μm.Bottom panel: Quantification of PNC prevalence reduction in HeLa cellstreated with the [IC_(50%)] (gray bar) or the [IC_(99%)] (black bar) ofeach compound (error bars=+SD). Amonafide, A1, and A4 are the onlycompounds that can significantly reduce PNC prevalence at their[IC_(50%)], while all the derivatives except A5 can significantly reducePNC prevalence at their [IC_(99%)].

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “subject is suspected of having cancer” refersto a subject that presents one or more symptoms indicative of a cancer(e.g., a noticeable lump or mass) or is being screened for a cancer(e.g., during a routine physical). A subject suspected of having cancermay also have one or more risk factors. A subject suspected of havingcancer has generally not been tested for cancer. However, a “subjectsuspected of having cancer” encompasses an individual who has received apreliminary diagnosis (e.g., a CT scan showing a mass) but for whom aconfirmatory test (e.g., biopsy and/or histology) has not been done orfor whom the stage of cancer is not known. The term further includespeople who once had cancer (e.g., an individual in remission). A“subject suspected of having cancer” is sometimes diagnosed with cancerand is sometimes found to not have cancer.

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention. A “preliminary diagnosis” is one based only on visual(e.g., CT scan or the presence of a lump) and antigen tests. As usedherein, the term “subject at risk for cancer” refers to a subject withone or more risk factors for developing a specific cancer. Risk factorsinclude, but are not limited to, gender, age, genetic predisposition,environmental expose, and previous incidents of cancer, preexistingnon-cancer diseases, and lifestyle.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, ayes, etc.

As used herein, the term “effective amount” refers to the amount of acomposition (e.g., comprising a compound of the present invention)sufficient to effect beneficial or desired results. An effective amountcan be administered in one or more administrations, applications ordosages and is not intended to be limited to a particular formulation oradministration route.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, or other agent, or therapeutic treatment (e.g.,compositions of the present invention) to a subject (e.g., a subject orin vivo, in vitro, or ex vivo cells, tissues, and organs). Exemplaryroutes of administration to the human body can be through the eyes(ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs(inhalant), oral mucosa (buccal), ear, by injection (e.g.,intravenously, subcutaneously, intratumorally, intraperitoneally, etc.)and the like.

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) (e.g., a compound of the presentinvention and an anti-cancer agent) or therapies to a subject. In someembodiments, the co-administration of two or more agents or therapies isconcurrent. In other embodiments, a first agent/therapy is administeredprior to a second agent/therapy. Those of skill in the art understandthat the formulations and/or routes of administration of the variousagents or therapies used may vary. The appropriate dosage forco-administration can be readily determined by one skilled in the art.In some embodiments, when agents or therapies are co-administered, therespective agents or therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents or therapies lowers the requisite dosage of a potentially harmful(e.g., toxic) agent(s).

As used herein, the term “toxic” refers to any detrimental or harmfuleffects on a subject, a cell, or a tissue as compared to the same cellor tissue prior to the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent (e.g., a compound of the presentinvention) with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vitro, in vivoor ex vivo.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable,” as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “topically” refers to application of thecompositions of the present invention to the surface of the skin andmucosal cells and tissues (e.g., alveolar, buccal, lingual, masticatory,or nasal mucosa, and other tissues and cells that line hollow organs orbody cavities).

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers including, but not limitedto, phosphate buffered saline solution, water, emulsions (e.g., such asan oil/water or water/oil emulsions), and various types of wettingagents, any and all solvents, dispersion media, coatings, sodium laurylsulfate, isotonic and absorption delaying agents, disintrigrants (e.g.,potato starch or sodium starch glycolate), and the like. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. (1975), incorporated herein by reference in its entirety).

As used herein, the term “pharmaceutically acceptable salt” refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound of the present invention that is physiologically tolerated inthe target subject (e.g., a mammalian subject, and/or in vivo or exvivo, cells, tissues, or organs). “Salts” of the compounds of thepresent invention may be derived from inorganic or organic acids andbases. Examples of acids include, but are not limited to, hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic,malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and thelike. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate,persulfate, phenylpropionate, picrate, pivalate, propionate, succinate,tartrate, thiocyanate, tosylate, undecanoate, and the like. Otherexamples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like. For therapeutic use,salts of the compounds of the present invention are contemplated asbeing pharmaceutically acceptable. However, salts of acids and basesthat are non-pharmaceutically acceptable may also find use, for example,in the preparation or purification of a pharmaceutically acceptablecompound.

As used herein, the terms “6-position amino derivative” refer to6-amino-2-[2-(dimethylamino)ethyl]1H-Benz[de]isoquinoline-1,3(2H)-diones.

As used herein, the terms “drug” and “chemotherapeutic agent” refer topharmacologically active molecules that are used to diagnose, treat, orprevent diseases or pathological conditions in a physiological system(e.g., a subject, or in vivo, in vitro, or ex vivo cells, tissues, andorgans). Drugs act by altering the physiology of a living organism,tissue, cell, or in vitro system to which the drug has beenadministered. It is intended that the terms “drug” and “chemotherapeuticagent” encompass anti-hyperproliferative and antineoplastic compounds aswell as other biologically therapeutic compounds.

The terms “analog” and “derivative” of a compound, as used herein,refers to a chemically modified compound wherein the chemicalmodification takes place either at a functional group of the compound,aromatic ring, or carbon backbone. Such derivatives include esters ofalcohol-containing compounds, esters of carboxy-containing compounds,amides of amine-containing compounds, amides of carboxy-containingcompounds, imines of amino-containing compounds, acetals ofaldehyde-containing compounds, ketals of carbonyl-containing compounds,and the like. The term “6-position amino derivative” specifically refersto6-amino-2-[2-(dimethylamino)ethyl]1H-Benz[de]isoquinoline-1,3(2H)-diones.

A “hyperproliferative disease,” as used herein refers to any conditionin which a localized population of proliferating cells in an animal isnot governed by the usual limitations of normal growth. Examples ofhyperproliferative disorders include tumors, neoplasms, lymphomas andthe like. A neoplasm is said to be benign if it does not undergoinvasion or metastasis and malignant if it does either of these. A“metastatic” cell or tissue means that the cell can invade and destroyneighboring body structures. Hyperplasia is a form of cell proliferationinvolving an increase in cell number in a tissue or organ withoutsignificant alteration in structure or function. Metaplasia is a form ofcontrolled cell growth in which one type of fully differentiated cellsubstitutes for another type of differentiated cell. Metaplasia canoccur in epithelial or connective tissue cells. A typical metaplasiainvolves a somewhat disorderly metaplastic epithelium.

As used herein, the term “neoplastic disease” refers to any abnormalgrowth of cells or tissues being either benign (non-cancerous) ormalignant (cancerous).

As used herein the term, “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes. The term “in vivo” refers to the natural environment(e.g. cell culture) and to processes or reactions that occur within anatural environment.

As used herein, the term “cell culture” refers to any culture of cells.Included within this term are continuous cell lines (e.g., with animmortal phenotype), primary cell cultures, finite cell lines (e.g.,non-transformed cells), and any other cell population maintained inculture, including oocytes and embryos.

DETAILED DESCRIPTION OF THE INVENTION

Amonafide is a DNA intercalator and topoisomerase II (topo II) inhibitor[1-3] that has shown good activity in several clinical trials [4-6]. Themalate salt of amonafide and the dihyrdochloride salt is currently underclinical development for the treatment of acute myelocytic leukemia [7]and the dihyrdochloride salt is under clinical development for thetreatment of refractory prostate carcinoma [8]. Amonafide is convertedto two main metabolites in humans, which are an inactive/non-toxicN′-hydroxylated metabolite and an active/toxic N-acetyl metabolite[9-13] (FIG. 1A). The latter is produced by N-acetyl transferase 2(NAT2) [12], which is a polymorphic enzyme causing differentialacetylation activity among individuals [13]. N-acetyl amonafide has beensuggested to compete for metabolic inactivation via CYP1A2 (FIG. 1A)with the parent drug causing increased plasma levels of active drug,hence causing toxicities [9]. Patients that are phenotyped as fastacetylators produce higher levels of the toxic N-acetylated metabolitecausing increased drug related toxicities compared to slow acetylators[14]. The extent of this differential acetylation causes some slowacetylators to be severely under dosed and some fast acetylators toexperience grade 4 toxicities at a fixed dose [9]. This problem hasforced physicians to determine patient's acetylator status based oncaffeine (a substrate for NAT2) acetylation rate [9, 15] or bygenotyping [8] in order to optimize the dose of amonafide for eachgroup. These phenotyping/genotyping assays delay treatment and add costto the treatment regimen. In addition, once phenotyped or genotyped,fast acetylators receive a lower dose of amonafide [15], which maydecrease the efficacy of amonafide treatment for this group of patients.

A derivative of amonafide that cannot be acetylated, but retainsbiological activity, may allow consistent treatment regimens forpatients independent of acetylator phenotype. This hypothesis has beenaddressed in previous studies with the creation of the amonafidederivatives, azonafide [16] and mitonafide [17]. Azonafide contains ananthracene ring instead of the naphthalene ring of amonafide, and has nofree aryl amine. Mitonafide has a nitro group in the 5-position, insteadof the free amine of amonafide. Both of these compounds avoid NAT2 basedmetabolism due a lack of an aryl amine and both are effectivelycytotoxic to cultured cancer cells [17-18]. Also, very potentbis-napthalamide derivatives, including elinafide, that lack an arylamine have been synthesized [19-20]; however, each of the derivativesmentioned is chemically much different from amonafide, which most likelyaffects the target, cellular uptake, and/or bioavailability of thedrugs. Therefore, there is still a need for an amonafide derivative thatis not able to be metabolized by NAT2, but is chemically similar toamonafide and retains the potency, selectivity, and biological activityof amonafide.

Experiments conducted during the course of development of embodimentsfor the present invention investigated derivatives of amonafide thatcannot be acetylated by NAT2, while maintaining biological activitiessimilar to (e.g., better than) amonafide. Such derivatives weresynthesized by moving the free aryl amine from the 5- to the 6-position,by adding functional groups to the 6-position amine, and/or bycompletely removing the aryl amine. Nine derivatives were synthesizedand the biological activities of each were directly compared withamonafide and other pertinent controls using purified systems and invitro assays. In particular, eight derivatives have aryl amines at the6-position (vs. 5-position of amonafide) and one derivative completelylacks the aryl amine. The derivative with a free amine in the 6-positionand one with a substituted amine in the 6-position are not acetylatedwhile amonafide is extensively acetylated as determined by an NAT2assay. The biological activities of these compounds were evaluated todetermine if they behaved similarly to amonafide in purified systems andin vitro. It was found that three compounds had similar cancercell-selective growth inhibition to amonafide, while also retainingsimilar subcellular localization, DNA intercalation and topoisomerase IIinhibition activities. In addition, these compounds were able toeliminate a marker of metastatic potential, the perinucleolarcompartment. Thus, these three compounds (named numonafides) allow forbetter patient management than those treated with amonafide and henceshould be developed further as potential clinical replacements foramonafide or as novel anti-cancer drugs.

Exemplary compositions and methods of the present invention aredescribed in more detail in the following sections: I. ExemplaryCompounds; II. Pharmaceutical compositions, formulations, and exemplaryadministration routes and dosing considerations; and III. TherapeuticApplications.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“Molecular cloning: a laboratory manual” Second Edition (Sambrook etal., 1989); “Oligonucleotide synthesis” (M. J. Gait, ed., 1984); “Animalcell culture” (R. I. Freshney, ed., 1987); the series “Methods inenzymology” (Academic Press, Inc.); “Handbook of experimentalimmunology” (D. M. Weir & C. C. Blackwell, eds.); “Gene transfer vectorsfor mammalian cells” (J. M. Miller & M. P. Calos, eds., 1987); “Currentprotocols in molecular biology” (F. M. Ausubel et al., eds., 1987, andperiodic updates); “PCR: the polymerase chain reaction” (Mullis et al.,eds., 1994); and “Current protocols in immunology” (J. E. Coligan etal., eds., 1991), each of which is herein incorporated by reference inits entirety.

I. Exemplary Compounds

Exemplary compounds of the present invention are provided below. Incertain embodiments, the present invention provides compounds describedby the following formulas:

including salts, esters, and prodrugs thereof; and including both R andS enantiomeric forms and racemic mixtures thereof; where X is present orabsent, and if present is

wherein R is H, ethyl, or R1, and wherein R1 is H, ethyl,

In certain embodiments, the present invention provides the followingcompounds:

II. Pharmaceutical Compositions, Formulations, and ExemplaryAdministration Routes and Dosing Considerations

Exemplary embodiments of various contemplated medicaments andpharmaceutical compositions are provided below.

A. Preparing Medicaments

It is contemplated that the compounds of the present invention areuseful in the preparation of medicaments to treat a variety ofconditions associated with cancer and/or hyperproliferation.

In addition, it is contemplated that the compounds are also useful forpreparing medicaments for treating other disorders wherein theeffectiveness of the compounds are known or predicted. Such disordersinclude, but are not limited to, neurological (e.g., epilepsy) orneuromuscular disorders. The methods and techniques for preparingmedicaments of a compound of the present invention are well-known in theart. Exemplary pharmaceutical formulations and routes of delivery aredescribed below.

One of skill in the art will appreciate that any one or more of thecompounds described herein, including the many specific embodiments, areprepared by applying standard pharmaceutical manufacturing procedures.Such medicaments can be delivered to the subject by using deliverymethods that are well-known in the pharmaceutical arts.

B. Exemplary Pharmaceutical Compositions and Formulation

In some embodiments of the present invention, the compositions areadministered alone, while in some other embodiments, the compositionsare preferably present in a pharmaceutical formulation comprising atleast one active ingredient/agent, as defined above, together with asolid support or alternatively, together with one or morepharmaceutically acceptable carriers and optionally other therapeuticagents. Each carrier must be “acceptable” in the sense that it iscompatible with the other ingredients of the formulation and notinjurious to the subject.

Contemplated formulations include those suitable oral, rectal, nasal,topical (including transdermal, buccal and sublingual), vaginal,parenteral (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary administration. In some embodiments,formulations are conveniently presented in unit dosage form and areprepared by any method known in the art of pharmacy. Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association (e.g., mixing) the active ingredient withliquid carriers or finely divided solid carriers or both, and then ifnecessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,wherein each preferably contains a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. In other embodiments, the activeingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more accessory agents/carriers are made by compressingor molding the respective agents. In some embodiments, compressedtablets are prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose)surface-active or dispersingagent. Molded tablets are made by molding in a suitable machine amixture of the powdered compound (e.g., active ingredient) moistenedwith an inert liquid diluent. Tablets may optionally be coated or scoredand may be formulated so as to provide slow or controlled release of theactive ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.Tablets may optionally be provided with an enteric coating, to providerelease in parts of the gut other than the stomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention are optionally formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. In alternatively embodiments, topical formulations comprisepatches or dressings such as a bandage or adhesive plasters impregnatedwith active ingredient(s), and optionally one or more excipients ordiluents. In some embodiments, the topical formulations include acompound(s) that enhances absorption or penetration of the activeagent(s) through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethylsulfoxide (DMSO) andrelated analogues.

If desired, the aqueous phase of a cream base includes, for example, atleast about 30% w/w of a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof.

In some embodiments, oily phase emulsions of this invention areconstituted from known ingredients in an known manner. This phasetypically comprises an lone emulsifier (otherwise known as an emulgent),it is also desirable in some embodiments for this phase to furthercomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier so as to act as a stabilizer. It some embodimentsit is also preferable to include both an oil and a fat. Together, theemulsifier(s) with or without stabilizer(s) make up the so-calledemulsifying wax, and the wax together with the oil and/or fat make upthe so-called emulsifying ointment base which forms the oily dispersedphase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired properties (e.g., cosmetic properties), since thesolubility of the active compound/agent in most oils likely to be usedin pharmaceutical emulsion formulations is very low. Thus creams shouldpreferably be a non-greasy, non-staining and washable products withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration may be presented as a suppositorywith suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, creams, gels, pastes, foams or spray formulations containingin addition to the agent, such carriers as are known in the art to beappropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include coarse powders having a particle size, for example, inthe range of about 20 to about 500 microns which are administered in themanner in which snuff is taken, i.e., by rapid inhalation (e.g., forced)through the nasal passage from a container of the powder held close upto the nose. Other suitable formulations wherein the carrier is a liquidfor administration include, but are not limited to, nasal sprays, drops,or aerosols by nebulizer, an include aqueous or oily solutions of theagents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. In some embodiments, the formulations arepresented/formulated in unit-dose or multi-dose sealed containers, forexample, ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents. It also is intended that the agents, compositions and methods ofthis invention be combined with other suitable compositions andtherapies. Still other formulations optionally include food additives(suitable sweeteners, flavorings, colorings, etc.), phytonutrients(e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, andother acceptable compositions (e.g., conjugated linoelic acid),extenders, and stabilizers, etc.

In some embodiments, the compounds of the present invention are providedin unsolvated form or are in non-aqueous solutions (e.g., ethanol). Thecompounds may be generated to allow such formulations through theproduction of specific crystalline polymorphs compatible with theformulations.

In certain embodiments, the present invention provides instructions foradministering said compound to a subject. In certain embodiments, thepresent invention provides instructions for using the compositionscontained in a kit for the treatment of conditions characterized by theconditions associated with cancer and/or hyperproliferation (e.g.,providing dosing, route of administration, decision trees for treatingphysicians for correlating patient-specific characteristics withtherapeutic courses of action). In certain embodiments, the presentinvention provides instructions for using the compositions contained inthe kit to treat cancer and/or hyperproliferative disorders (e.g.,tumors, B cell lymphomas, T cell lymphomas, etc.).

C. Exemplary Administration Routes and Dosing Considerations

Various delivery systems are known and can be used to administertherapeutic agents (e.g., exemplary compounds as described in Section Iabove) of the present invention, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor-mediated endocytosis, and thelike. Methods of delivery include, but are not limited to,intra-arterial, intra-muscular, intravenous, intranasal, and oralroutes. In specific embodiments, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, injection, or by means of acatheter.

It is contemplated that the agents identified can be administered tosubjects or individuals susceptible to or at risk of developingpathological growth of target cells and correlated conditions. When theagent is administered to a subject such as a mouse, a rat or a humanpatient, the agent can be added to a pharmaceutically acceptable carrierand systemically or topically administered to the subject. To determinepatients that can be beneficially treated, a tissue sample is removedfrom the patient and the cells are assayed for sensitivity to the agent.

Therapeutic amounts are empirically determined and vary with thepathology being treated, the subject being treated and the efficacy andtoxicity of the agent.

In some embodiments, in vivo administration is effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are well known to those of skill in the art and vary withthe composition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations are carried out with the dose level and pattern beingselected by the treating physician.

Suitable dosage formulations and methods of administering the agents arereadily determined by those of skill in the art. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. When the compounds describedherein are co-administered with another agent (e.g., as sensitizingagents), the effective amount may be less than when the agent is usedalone.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor nonaqueous diluents, syrups, granulates or powders. In addition to anagent of the present invention, the pharmaceutical compositions can alsocontain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

More particularly, an agent of the present invention also referred toherein as the active ingredient, may be administered for therapy by anysuitable route including, but not limited to, oral, rectal, nasal,topical (including, but not limited to, transdermal, aerosol, buccal andsublingual), vaginal, parental (including, but not limited to,subcutaneous, intramuscular, intravenous and intradermal) and pulmonary.It is also appreciated that the preferred route varies with thecondition and age of the recipient, and the disease being treated.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of disease. This may be achieved, forexample, by the intravenous injection of the agent, optionally insaline, or orally administered, for example, as a tablet, capsule orsyrup containing the active ingredient.

Desirable blood levels of the agent may be maintained by a continuousinfusion to provide a therapeutic amount of the active ingredient withindisease tissue. The use of operative combinations is contemplated toprovide therapeutic combinations requiring a lower total dosage of eachcomponent antiviral agent than may be required when each individualtherapeutic compound or drug is used alone, thereby reducing adverseeffects.

D. Exemplary Co-Administration routes and dosing considerations

The present invention also includes methods involving co-administrationof the compounds described herein with one or more additional activeagents. Indeed, it is a further aspect of this invention to providemethods for enhancing prior art therapies and/or pharmaceuticalcompositions by co-administering a compound of this invention. Inco-administration procedures, the agents may be administeredconcurrently or sequentially. In one embodiment, the compounds describedherein are administered prior to the other active agent(s). Thepharmaceutical formulations and modes of administration may be any ofthose described above. In addition, the two or more co-administeredchemical agents, biological agents or radiation may each be administeredusing different modes or different formulations.

The agent or agents to be co-administered depends on the type ofcondition being treated. For example, when the condition being treatedis cancer, the additional agent can be a chemotherapeutic agent orradiation. The additional agents to be co-administered and can be any ofthe well-known agents in the art, including, but not limited to, thosethat are currently in clinical use. The determination of appropriatetype and dosage of radiation treatment is also within the skill in theart or can be determined with relative ease.

The present invention is not limited by type of anti-cancer agentco-administered. Indeed, a variety of anti-cancer agents arecontemplated to be useful in the present invention including, but notlimited to, Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;Adozelesin; Adriamycin; Aldesleukin; Alitretinoin; Allopurinol Sodium;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Annonaceous Acetogenins; Anthramycin; Asimicin;Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat;Benzodepa; Bexarotene; Bicalutamide; Bisantrene Hydrochloride; BisnafideDimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine;Bullatacin; Busulfan; Cabergoline; Cactinomycin; Calusterone;Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Celecoxib; Chlorambucil;Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; DACA(N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin;Daunorubicin Hydrochloride; Daunomycin; Decitabine; Denileukin Diftitox;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized OilI 131; Etoposide; Etoposide Phosphate; Etoprine; FadrozoleHydrochloride; Fazarabine; Fenretinide; Floxuridine; FludarabinePhosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; FostriecinSodium; FK-317; FK-973; FR-66979; FR-900482; Gemcitabine; GeimcitabineHydrochloride; Gemtuzumab Ozogamicin; Gold Au 198; Goserelin Acetate;Guanacone; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-1a; Interferon Gamma-1b; Iproplatin;Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; LeuprolideAcetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine;Losoxantrone Hydrochloride; Masoprocol; Maytansine; MechlorethamineHydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Methoxsalen; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;Mitogillin; Mitomalcin; Mitomycin; Mytomycin C; Mitosper; Mitotane;Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;Oprelvekin; Ormaplatin; Oxisuran; Paclitaxel; Pamidronate Disodium;Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rituximab; Rogletimide; Rolliniastatin;Safingol; Safingol Hydrochloride; Samarium/Lexidronam; Semustine;Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Squamocin; Squamotacin;Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur;Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine;Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene Citrate;Trastuzumab; Trestolone Acetate; Triciribine Phosphate; Trimetrexate;Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; UracilMustard; Uredepa; Valrubicin; Vapreotide; Verteporfin; Vinblastine;Vinblastine Sulfate; Vincristine; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride; 2-Chlorodeoxyadenosine; 2′-Deoxyformycin;9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid;2-chloro-2′-arabino-fluoro-2′-deoxyadenosine;2-chloro-2′-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R;CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlorethamine);cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan;N-methyl-N-nitrosourea (MNU); N,N′-Bis(2-chloroethyl)-N-nitrosourea(BCNU); N-(2-chloroethyl)-N′-cyclohex-yl-N-nitrosourea (CCNU);N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU);N-(2-chloroethyl)-N′-(diethyl)ethylphosphonate-N-nit-rosourea(fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide;temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin;Carboplatin; Ormaplatin; Oxaliplatin; C1-973; DWA 2114R; JM216; JM335;Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine;6-Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide; 9-aminocamptothecin; Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin;darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);amsacrine; pyrazoloacridine; all-trans retinol;14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-Hydroxyphenyl)retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid;fludarabine (2-F-ara-AMP); and 2-chlorodeoxyadenosine (2-Cda).

Other anti-cancer agents include: Antiproliferative agents (e.g.,Piritrexim Isothionate), Antiprostatic hypertrophy agent (e.g.,Sitogluside), Benign prostatic hyperplasia therapy agents (e.g.,Tamsulosin Hydrochloride), Prostate growth inhibitor agents (e.g.,Pentomone), and Radioactive agents: Fibrinogen 1 125; Fludeoxyglucose F18; Fluorodopa F 18; Insulin I 125; Insulin I 131; Iobenguane I 123;Iodipamide Sodium I 131; Iodoantipyrine I 131; Iodocholesterol I 131;Iodohippurate Sodium I 123; Iodohippurate Sodium I 125; IodohippurateSodium I 131; Iodopyracet I 125; Iodopyracet I 131; IofetamineHydrochloride I 123; Iomethin I 125; Iomethin I 131; Iothalamate SodiumI 125; Iothalamate Sodium I 131; Iotyrosine I 131; Liothyronine I 125;Liothyronine I 131; Merisoprol Acetate Hg 197; Merisoprol Acetate Hg203; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Tc 99mAntimony Trisulfide Colloid; Technetium Tc 99m Bicisate; Technetium Tc99m Disofenin; Technetium Tc 99m Etidronate; Technetium Tc 99mExametazime; Technetium Tc 99m Furifosmin; Technetium Tc 99m Gluceptate;Technetium Tc 99m Lidofenin; Technetium Tc 99m Mebrofenin; Technetium Tc99m Medronate; Technetium Tc 99m Medronate Disodium; Technetium Tc 99mMertiatide; Technetium Tc 99m Oxidronate; Technetium Tc 99m Pentetate;Technetium Tc 99m Pentetate Calcium Trisodium; Technetium Tc 99mSestamibi; Technetium Tc 99m Siboroxime; Technetium Tc 99m Succimer;Technetium Tc 99m Sulfur Colloid; Technetium Tc 99m Teboroxime;Technetium Tc 99m Tetrofosmin; Technetium Tc 99m Tiatide; Thyroxine I125; Thyroxine I 131; Tolpovidone I 131; Triolein I 125; Triolein I 131.

Another category of anti-cancer agents is anti-cancer SupplementaryPotentiating Agents, including: Tricyclic anti-depressant drugs (e.g.,imipramine, desipramine, amitryptyline, clomipramine, trimipramine,doxepin, nortriptyline, protriptyline, amoxapine and maprotiline);non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone andcitalopram); Ca⁺⁺ antagonists (e.g., verapamil, nifedipine, nitrendipineand caroverine); Calmodulin inhibitors (e.g., prenylamine,trifluoroperazine and clomipramine); Amphotericin B; Triparanolanalogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine);antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g.,buthionine and sulfoximine) and Multiple Drug Resistance reducing agentssuch as Cremaphor EL.

Still other anticancer agents are those selected from the groupconsisting of: annonaceous acetogenins; asimicin; rolliniastatin;guanacone, squamocin, bullatacin; squamotacin; taxanes; paclitaxel;gemcitabine; methotrexate FR-900482; FK-973; FR-66979; FK-317; 5-FU;FUDR; FdUMP; Hydroxyurea; Docetaxel; discodermolide; epothilones;vincristine; vinblastine; vinorelbine; meta-pac; irinotecan; SN-38;10-OH campto; topotecan; etoposide; adriamycin; flavopiridol; Cis-Pt;carbo-Pt; bleomycin; mitomycin C; mithramycin; capecitabine; cytarabine;2-C1-2′deoxyadenosine; Fludarabine-PO₄; mitoxantrone; mitozolomide;Pentostatin; and Tomudex.

One particularly preferred class of anticancer agents are taxanes (e.g.,paclitaxel and docetaxel). Another important category of anticanceragent is annonaceous acetogenin.

Other cancer therapies include hormonal manipulation. In someembodiments, the anti-cancer agent is tamoxifen or the aromataseinhibitor arimidex (i.e., anastrozole).

III. Therapeutic Application

In certain embodiments, the present invention provides methods (e.g.,therapeutic applications) for regulating treating conditions associatedwith cancer and/or hyperproliferation comprising: a) providing: i. asubject (e.g., a human subject diagnosed with cancer) diagnosed withcancer; and ii. a composition (e.g., exemplary compounds as described inSection III above); and b) exposing the subject to the composition underconditions such that the exposure results in cancer cell death. Thepresent invention is not limited to a particular therapeuticapplication. Non-limiting examples of therapeutic applications for thepresent invention are described in the following subsections. Thepresent invention is not limited to treating particular types of cancer.Examples of cancer include, but are not limited to, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, bladder cancer, leukemia cancer, prostate cancer, renalcancer, uterine cancer, ovarian cancer, breast cancer, colon cancer,cervical cancer, and lung cancer. In some embodiments, the compositionsof the present invention are co-administered with one or more additionaltreatment agents (e.g., anti-cancer agents described in Section II).

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof

Methods Chemical Synthesis

A total of eight amonafide derivatives with amines in the 6-positionwere synthesized (A1 to A8) as well as a derivative completely lackingan aryl amine (A9). The synthesis scheme is shown in FIG. 1B and thesynthesis details are listed below. A1 and A9 have been synthesizedpreviously [21, 17]. Amonafide was obtained from the National CancerInstitute's Developmental Therapeutics Program (Bethesda, Md.). Allcompounds were stored at 10 mM in DMSO at −20° C.

Analysis of synthetic products: Proton nuclear magnetic resonances (¹HNMR) were recorded in deuterated solvents on a Gemini 300 (300 MHz) oriNOVA 500 (500 MHz) spectrometer. Chemical shifts are reported in partsper million (ppm, δ) relative to tetramethylsilane (δ 0.00). Iftetramethylsilane was not present, the residual protio solvent isreferenced (CDCl₃, δ 7.27; dimethylsulfoxide-d₆ (DMSO-d₆), δ 2.50). ¹HNMR splitting patterns are designated as singlet (s), doublet (d),triplet (t), or quartet (q). Splitting patterns that could not beinterpreted or easily visualized were designated as multiplet (m) orbroad (br). In some cases, the signals from exchangeable protons werenot able to be identified. Coupling constants are reported in Hertz(Hz). Mass spectra were obtained using an API 3000 LC/MS/MS system, aMicromass Quattro II Triple Quadrupole HPLC/MS/MS Mass Spectrometer, ora Waters LCT Premier time-of-flight (TOF) mass spectrometer. Analyticalthin layer chromatography (TLC) was carried out on Sorbent TechnologiesTLC plates precoated with silica gel (250 μm layer thickness). Flashcolumn chromatography was performed on EM Science silica gel 60 (230-400mesh). All commercially available reagents and solvents were purchasedfrom Aldrich (St. Louis, Mo.) and used without further purificationexcept for dimethylformamide (DMF), which was purified by passagethrough a bed of activated alumina.

Synthesis of 6-nitro-imide precursor(6-nitro-2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione):6-nitro-naphthylic anhydride (1.00 g, 4.11 mmol) was dissolved in DMF(40 mL) and the solution was cooled to 0° C. and N,N-dimethylethylenediamine (0.45 mL, 4.11 mmol) was added dropwise. The solution wasallowed to warm to room temperature and stirred for 24 hours. Thesolvent was removed under vacuum and purified by flash columnchromatography to yield 1.24 g (96%)4-nitro-N-(dimethylaminoethyl)naphthylic imide as a light brown solid.¹H NMR (500 MHz, CDCl₃) δ 8.84 (d, J=8.0 Hz, 1H, naphthylic-H) 8.40 (d,J=7.5 Hz, 1H, naphthylic-H) 8.69 (d, J=8.0 Hz, 1H, naphthylic-H) 8.40(d, J=8.0 Hz, 1H, naphthylic-H) 7.99 (t, J=8.0 Hz, 1H, naphthylic-H)4.34 (t, J=7.0 Hz, 2H, ethylene-CH₂) 2.67 (t, J=7.0 Hz, 2H,ethylene-CH₂) 2.34 (s, 6H, N(CH₃)₂). Calculated mass for [M+H]⁺=314.11;observed=314.2.

Synthesis of A1(6-amino-2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione):Nitro-N-(dimethylaminoethyl)naphthalic imide (200 mg, 0.64 mmol) wasdissolved in 95% ethanol and slowly added to a Parr flask containing 10%Pd/C. The mixture was placed on a Parr apparatus under 40 psi H₂pressure for 12 hours. The mixture was filtered through Celite and thesolvent was removed under vacuum.6-nitro-N-(dimethylaminoethyl)naphthalic imide was reduced viahydrogenolysis to yield 185 mg (99%) A1 as a bright orange solid. ¹H NMR(300 MHz, CDCl₃) δ 8.57 (d, J=7.0 Hz, 1H, naphthylic-H) 8.34 (d, J=8.0Hz, 1H, naphthylic-H) 8.05 (d, J=8.5 Hz, 1H, naphthylic-H) 7.62 (t, 1H,naphthylic-H) 6.79 (d, J=8.0 Hz, 1H, naphthylic-H) 5.04 (br s, 2H,amino-H) 4.32 (t, J=6.5 Hz, 2H, ethylene-CH₂) 2.68 (t, J=6.5 Hz, 2H,ethylene-CH₂) 2.38 (s, 6H, N(CH₃)₂). Calculated mass for [M+H]⁺=284.14;observed=284.14.

Synthesis of A2(6-propylamino-2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione):6-nitro-imide precursor (1.00 g, 3.19 mmol) was added to a high-pressurevessel and the solid was suspended in DMF (2 mL). Excess propylamine(1.0 mL) was added turning the suspension dark brown. The vessel wastightly sealed and heated to 100° C. for one hour. The solution wascooled to room temperature and the solvents were removed under vacuum.The dark brown residue was purified by flash column chromatography (5%MeOH/CH₂Cl₂) to yield 548 mg (53%) of A2 as a bright orange solid. Thisprocedure was used for A3-A8 as well. ¹H NMR (500 MHz, CDCl₃) δ 8.58 (d,J=7.0 Hz, 1H, naphthylic-H) 8.47 (d, J=8.5 Hz, 1H, naphthylic-H) 8.08(d, J=8.5 Hz, 1H, naphthylic-H) 7.62 (t, J=8.0 Hz, 1H, naphthylic-H)6.73 (d, J=8.5 Hz, 1H, naphthylic-H) 5.24 (br s, 1H, amino-H) 4.33 (t,J=7.0 Hz, 2H, ethylene-CH₂) 3.39 (m, 2H, NHCH₂CH₂CH₃) 2.69 (t, J=7.0 Hz,2H, ethylene-CH₂) 2.40 (s, 6H, N(CH₃)₂) 1.85 (m, 2H, NHCH₂CH₂CH₃) 1.12(t, J=7.0 Hz, 3H, NHCH₂CH₂CH₃). Calculated mass for [M+H]⁺=326.19;observed=326.1.

Synthesis of A3(6-allylamino-2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione):6-nitro-imide precursor (200 mg, 0.64 mmol) and excess allylamine (0.5mL) in DMF (1 mL), were reacted to yield 89.4 mg (43%) of A3 as a brightorange solid. ¹H NMR (300 MHz, CDCl₃) δ 8.59 (dd, J=7.2 Hz, 1H,naphthylic-H) 8.46 (d, J=8.4 Hz, 1H, naphthylic-H) 8.10 (dd, J=7.8 Hz,1H, naphthylic-H) 7.63 (dd, J=7.8 Hz, 1H, naphthylic-H) 6.73 (d, J=8.4Hz, 1H, naphthylic-H) 6.10-6.00 (ddt, 1H, CH₂CHCH₂) 5.45-5.30 (m, 2H,terminal alkene-H) 4.32 (t, J=7.2 Hz, 2H, ethylene-CH₂) 3.39 (m, 2H,allylic-H) 2.65 (t, J=7.2 Hz, 2H, ethylene-CH₂) 2.40 (s, 6H, N(CH₃)₂).Calculated mass for [M+H]⁺=324.40; observed=324.5.

Synthesis of A4(6-methoxyethylamino-2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione):6-nitro-imide precursor (200 mg, 0.64 mmol) and excess2-methoxyethylamine (0.5 mL) in DMF (1 mL) were reacted to yield 33.8 mg(15.5%) of A4 as a bright orange solid. ¹H NMR (300 MHz, CDCl₃) δ 8.59(d, J=7.5 Hz, 1H, naphthylic-H) 8.46 (d, J=8.7 Hz, 1H, naphthylic-H)8.13 (d, J=7.5 Hz, 1H, naphthylic-H) 7.63 (t, J=7.8 Hz, 1H,naphthylic-H) 6.72 (d, J=8.7 Hz, 1H, naphthylic-H) 5.66 (br m, 1H,amino-H) 4.32 (t, J=7.2 Hz, 2H, ethylene-CH₂) 3.78 (m, J=5.4 Hz, 2H,NHCH₂CH₂OCH₃) 3.58 (m, J=5.4 Hz, 2H, NHCH₂CH₂OCH₃) 3.47 (s, 3H,NHCH₂CH₂OCH₃) 2.65 (t, J=7.2 Hz, 2H, ethylene-CH₂) 2.37 (s, 6H,N(CH₃)₂). Calculated mass for [M+H]⁺=342.18; observed =342.18.

Synthesis of A5(6-hexylamino-2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione):6-nitro-imide precursor (200 mg, 0.64 mmol) and excess hexylamine (0.5mL) in DMF (1 mL) were reacted to yield 81.0 mg (34%) of A5 as a brightorange solid. ¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, J=7.5 Hz, 1H,naphthylic-H) 8.45 (d, J=8.4 Hz, 1H, naphthylic-H) 8.07 (d, J=8.7 Hz,1H, naphthylic-H) 7.61 (t, J=7.5 Hz, 1H, naphthylic-H) 6.71 (d, J=8.4Hz, 1H, naphthylic-H) 5.25 (br t, 1H, amino-H) 4.32 (t, J=7.2 Hz, 2H,ethylene-CH₂) 3.40 (q, J=7.2 Hz, 2H, NHCH₂(CH₂)₄CH₃) 2.66 (t, J=6.9 Hz,2H, ethylene-CH₂) 2.38 (s, 6H, N(CH₃)₂) 1.81 (m, 2H, hexyl-H) 1.55-1.35(m, 6H, hexyl-H) 0.93 (t, J=6.9 Hz, 3H, NHCH₂(CH₂)₄CH₃). Calculated massfor [M+H]⁺=368.50; observed=368.0.\

Synthesis of A6(6-cyclohexylamino-2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione):6-nitro-imide precursor (200 mg, 0.64 mmol) and excess cyclohexylamine(0.5 mL) in DMF (1 mL) were reacted to yield 104 mg (45%) of A6 as abright orange solid. ¹H NMR (300 MHz, CDCl₃) δ 8.58 (d, J=7.2 Hz, 1H,naphthylic-H) 8.44 (d, J=8.4 Hz, 1H, naphthylic-H) 8.05 (d, J=8.4 Hz,1H, naphthylic-H) 7.60 (t, J=8.4 Hz, 1H, naphthylic-H) 6.74 (d, J=8.7Hz, 1H, naphthylic-H) 5.16 (br d, 1H, amino-H) 4.31 (t, J=7.5 Hz, 2H,ethylene-CH₂) 3.62 (m, 1H, NHCH-cyclohexyl) 2.65 (t, J=6.3 Hz, 2H,ethylene-CH₂) 2.37 (s, 6H, N(CH₃)₂) 2.19 (m, 2H, cyclohexyl-CH₂)1.90-1.30 (m, 8H, cyclohexyl-CH₂). Calculated mass for [M+H]⁺=366.49;observed=366.0.

Synthesis of A7(6-piperdinyl-2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione):6-nitro-imide precursor (200 mg, 0.64 mmol) and excess piperidine (0.5mL) in DMF (1 mL) were reacted to yield 204 mg (91%) of A7 as a brightorange solid. ¹H NMR (300 MHz, CDCl₃) δ 8.57 (dd, J=7.2 Hz, 1H,naphthylic-H) 8.49 (d, J=8.1 Hz, 1H, naphthylic-H) 8.39 (dd, J=8.4 Hz,1H, naphthylic-H) 7.67 (dd, J=8.4 Hz, 1H, naphthylic-H) 7.17 (d, J=8.1Hz, 1H, naphthylic-H) 4.32 (t, J=7.2 Hz, 2H, ethylene-CH₂) 3.23 (m, 2H,piperdinyl-H) 2.65 (t, J=7.2 Hz, 2H, ethylene-CH₂) 2.36 (s, 6H, N(CH₃)₂)1.90-1.60 (m, 6H, piperdinyl-H). Calculate mass for [M+H]⁻=352.20;observed=352.20.

Synthesis of A8(6-diethylamino-2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione):6-nitro-imide precursor (200 mg, 0.64 mmol) was treated with excessdiethylamine (0.5 mL) in DMF (1 mL) to yield 55.6 mg (26%) of A8 as abright orange solid. ¹H NMR (300 MHz, CDCl₃) δ 8.58 (dd, J=7.2 Hz, 1H,naphthylic-H) 8.49 (d, J=8.4 Hz, 1H, naphthylic-H) 8.45 (dd, J=8.4 Hz,1H, naphthylic-H) 7.65 (dd, J=8.4 Hz, 1H, naphthylic-H) 7.21 (d, J=8.1Hz, 1H, naphthylic-H) 4.32 (t, J=7.0 Hz, 2H, ethylene-CH₂) 3.41 (q,J=7.2 Hz, 4H, N(CH₂CH₃)₂) 2.66 (t, J=7.0 Hz, 2H, ethylene-CH₂) 2.37 (s,6H, N(CH₃)₂) 1.68 (t, J=7.2 Hz, 6H, N(CH₂CH₃)₂). Calculated mass for[M+H]⁺=340.44; observed=340.1.

Synthesis of A9(2-[2-(dimethylamino)ethyl]-1H-benz[de]isoquinoline-1,3(2H)-dione): Thiscompound was synthesized according to previously published protocols andsimilar yields and spectral data were obtained [17]. Calculated mass for[M+H]⁺=269.32; observed=269.3.

NAT2 Assay: This assay was performed using a wild type recombinant humanNAT2 (NAT2*4) that was purified from E. coli as previously described[22]. The volume of each reaction was 500 μL with 200 μM amonafide, A1,or A2, 100 μg NAT2*4, and 400 μM AcetylCoA (Sigma—Poole, UK). Reactionswere run for 0 or 10 min and stopped by addition of 500 μL of 20% (w/v)trichloroacetic acid (TCA-Sigma-Poole, UK) to give a final TCAconcentration of 400 μM which precipitates the enzyme. Two controlassays, lacking either enzyme or compound, were performed for eachcompound. The reaction mixtures were prepared for analysis by isolationof the compounds from the reaction matrix by solid-phase extraction. Inbrief, 100 μL of the reaction mixture was added to 1 mL of 2% aceticacid. The sorbent of an Oasis HLB 30 mg Extraction Cartridge (WatersChromatography—Milford, Mass.) was conditioned with 1 mL of methanol and1 mL of water, the prepared sample was applied to the conditionedsorbent, and the sorbent was washed with 1 mL of 5% methanol. Sampleswere eluted with 200 μL methanol followed by 800 μL of 0.1% formic acidin methanol. The eluant was analyzed by an API 3000 LC-MS/MS system(Applied Biosystems—Foster City, Calif.). Samples were infused at 5μL/min directly into the TurboIonSpray Source (Applied Biosystems—FosterCity, Calif.), which was operated in the positive ionization mode. Theinfusions of all samples were subjected to Q1 scans from 150-600 m/z AMUwith unit resolution. Data are represented as the average ratio of theacetylated compound to the parent compound from three separateinjections. Reactions with no compound were extracted and injected aswell to ensure that there were no peaks in the reaction mixture thatcorresponded to the masses of amonafide (284.1m/z), A1 (284.3m/z) , A2(326.6m/z), or the acetylated forms (M+42.5m/z) thereof.

Cell Culture: HeLa (cervical carcinoma), WI-38 (normal human skinfibroblasts), and MDA-MB-231 (breast carcinoma), cells were cultured inDMEM (Invitrogen—Carlsbad, Calif.) and PC-3M (metastatic prostatecarcinoma) cells were cultured in RPMI-1640. Peripheral bloodmononuclear cells (PBMCs) were voluntarily collected from a healthylaboratory worker by standard sterile phlebotomy techniques and isolatedusing Vacutainer CPT collection tubes (BD Biosciences—Bedford, Mass.).PBMCs were isolated freshly for each experiment. All DMEM and RPMI-1640media was supplemented with 10% FBS (Atlanta Biologicals—Lawrenceville,Ga.) and 100 U/mL penicillin and streptomycin (Invitrogen). HUVEC (Humanumbilical vein endothelial cells) were cultured in EGM-2 media withsupplements provided in the manufacturers BulletKit (Lonza—Walersville,Md.). All cells were maintained at 37° C. and in a 5% CO₂ atmosphere.

Growth Inhibition Assays: HeLa, MDA-MB-231, PC-3M, WI-38, and HUVECcells were plated at 5,000 cells per well in 96 well plates and allowedto attach for 20 hr (8 hr for HeLa). The media was removed and 198 μL offresh media was added to each well. Each well was treated with one of 14doses for each compound (10⁵ nM, 10^(4.75) nM, 10^(4.5) nM, . . .10^(1.75) nM) for HeLa cells and on of 8 doses for each compound (10⁵nM, 10^(4.66) nM, 10^(4.33) nM, . . . 10^(2.66) nM) for the other celllines to give a final vehicle concentration of 1% DMSO and media volumeof 200 ρL. PBMCs were isolated and plated at 100,000 per well in 96 wellplates with a volume of 197 μL. They were allowed to settle in the mediafor 20 hr, at which point 1 μL of 1 mg/mL phytohemagglutinin (Sigma—St.Louis Mo.) in PBS was added to stimulate proliferation (finalconcentration of 5 μg/mL). Each well was treated with one of 8 doses foreach compound (10⁵nM, 10^(4.66) nM, 10^(4.33) nM, . . . 10^(2.66) nM) togive a final vehicle concentration of 1% DMSO and media volume of 200μL. Cell proliferation was measured at t=0 (time at start of treatment)and t=72 hr using MTS AQueous non-radioactive proliferation assay(Promega—Madison, Wis.). This assay utilizes the metabolic conversion ofyellow tetrazolium to a purple formazan by live cells and the colorchange is proportional to the number of cells [23]. For HeLa,MDA-MB-231, PC-3M, WI-38, HUVEC, cells, the media was removed andreplaced with 120 μL of 1:5 v/v MTS:media solution and allowed toincubate for 1 hr at 37° C. For PBMCs, 40 μL of the MTS solution wasadded directly to the media and allowed to incubate for 4 hr at 37° C.The absorbance at 490 nm in each well was read with a Spectramax 250plate reader (Molecular Devices—Sunnyvale, Calif.). The absorbance att=0 was subtracted from all absorbances at t=72 hr so that a 100% growthinhibitory concentration ([GI_(100%)]) corresponds to whenabs_(72hr)=abs_(0hr). Each experiment was repeated four times and thedata for each concentration were averaged and growth inhibition curveswere constructed using XLfit4 software (IDBS—Guildford, UK). Data werefit to one site dose response curves in order to obtain specific growthinhibitory concentrations and the relative standard errors of the meanfor these concentrations were calculated by the XLfit4 software.Statistical significance (p value) for the selectivity and allsubsequent assays was determined with the two tailed homoscedasticStudent's t-test. Subsequent assays that utilize the [GI_(99%)] are 20hr in duration and assays that utilize the [GI_(50%)] are 72 hr induration. GI doses are used in subsequent assays to standardize theeffects of the compounds, so that biological responses can be correlatedamong the different compounds at equitoxic doses.

Subcellular Compound Localization: HeLa cells were plated at 250,000 per35 mm well with a sterile glass coverslip. The cells were allowed toattach for 20 hr and then treated with the [GI_(99%)] of each derivative20 hr (total media volume 10 mL/well). The media was removed and thecells were rapidly fixed in 4% w/v paraformaldehyde in PBS for 12 min,permeabilized with 0.5% w/v Triton X-100 in PBS for 5 min, stained with1.5 mU/μL Texas Red labeled phalloidin (Invitrogen) for 15 min to markthe cytoplasm (F-actin), counterstained with 50 ng/mL DAPI in PBS for 2min to mark the nuclei (heterochromatin), and then mounted on slideswith mounting media (Vector Laboratories—Burlingame, Calif.). Thefluorescence of the compounds was detectable in the FITC/GFP channel(490 nm-520 nm emission filter) of a Nikon E800 fluorescent microscope.Images are representative for each compound and were acquired using a100× objective, SenSys cooled CCD camera (Photometrics—Tucson, Ariz.),and MetaView v 4.5 software (Universal Imaging Corp.—West Chester, Pa.).Scale bars=10 μm. DAPI staining was used to bring the nucleus, includingthe nucleoli, into focus and this focus plane was used to acquire imagesin the blue and green channels. The microscope was refocused at thebottom of the cell to acquire images of the phalloidin staining todefinitively mark the cytoplasmic boundaries. Images of vehicle treatedcells were acquired with the same exposure, brightness, and contrast ascompound treated cells to ensure that the signals in the green channelare not due to auto fluorescence of the cells. The vehicle image in FIG.3 is representative of these images. A9 does not fluoresce, hence wasexcluded from this assay.

DNA Intercalation Assay: A DNA unwinding assay was performed aspreviously described [1] with minor modifications. Four Units ofrecombinant wild type human topoisomerase I (Topogen—Port Orange, Fla.)were incubated with reaction buffer (included with enzyme), ˜0.5 μg ofpBR322 plasmid DNA, which is a mixture of form I and form II plasmidDNA, and water added to a final volume of 19 μL. The reactions wereincubated at 37° C. for 30 min and then 1 μL of each compound was addedto give a final concentration of 10, 31.6, and 100 μM (final solventconcentration of 1% DMSO) and the reaction mixture was incubated at 37°C. for another 30 min. The reaction was stopped by the addition of 5 μLgel loading dye [1]. The samples were immediately loaded onto a 1%agarose gel and electrophoresis was performed at 50 V for 3 h at roomtemperature. Pictures of the gels were obtained using a Kodak ImageStation 440 CF (Kodak—Rochester, N.Y.) equipped with a UV source.

In vitro Topoisomerase Inhibition: HeLa cells were plated at a densityof 1.5 million in 35 mm wells and allowed to attach for 20 hr. They werethen treated with each compound at the [GI_(99%)] in 10 mL media for 20hr. In vitro topoisomerase II inhibition was quantified using TopoGEN'sTopoisomerase II In vivo Link Kit according to the protocol includedwith the kit and according to a previously published protocol [24] withslight modifications. In summary, DNA was isolated from sarkosyl lysedcells, layered on a cesium chloride gradient (total volume ˜7 mL),ultra-centrifuged, and then fractionated to 0.5 mL. The fractions werediluted 1:4 v/v in PBS and the fractions containing the genomic DNA weredetermined by measuring the absorbance at 260 nm for each fraction. Thefractions were then loaded into a slot blotting apparatus and pulledthrough a nitrocellulose membrane by vacuum. The membrane was blocked in5% w/v milk in 1× TBST (blotto) for 2 hr, incubated in 1:5,000 v/vtopoisomerase II (topo II) primary antibody (TopoGEN) in blotto for 1hr, washed, and incubated in 1:2,000 v/v HRP conjugated anti-rabbitsecondary antibody (Jackson—West Grove, Pa.) in blotto for 45 min,washed and developed with SuperSignal West Pico ChemiluminescentSubstrate (Pierce—Rockford, Ill.). The data were processed by dividingthe intensity of the 2 or 3 most intense topo II immuno-reactive bandsby the amount of DNA in the respective fractions. The band intensity wasdetermined by scanning the developed films with a Kodak Image Station440 CF and measuring the average pixel intensity of an area slightlylarger than each band and subtracting the median background pixelintensity of the perimeter of the same area using Kodak MI software.Etoposide (TopoGEN) and mitoxantrone (Sigma—St. Louis, Mo.) are topo IIinhibitors used as positive controls. Camptothecin (Sigma—St. Louis,Mo.) is a selective topoisomerase I inhibitor and was used as negativecontrol. Data were standardized to mitoxantrone in each of threeindependent experiments.

DNA Damage Response Assay: 250,000 HeLa cells were plated in 35 mmculture dishes and allowed to attach for 8 hr and were then treated withthe [GI_(99%)] of each compound for 20 hr (total media volume was 10mL/well with 1% DMSO). After treatment cells were lysed in 150 μLdetergent buffer containing 1:100 v/v protease inhibitor cocktail(Sigma—St. Louis, Mo.). Lysates were run on denaturing 12% acrylamidegels and transferred to nitrocellulose membranes. The membranes wereblocked in blotto, incubated in anti-pThr68-Chk2 antibody (CellSignaling—Danvers, Mass.) at 1:500 v/v dilution in blotto overnight 4°C., washed, incubated in HRP conjugated anti-rabbit secondary antibody(Jackson) at 1:5,000 v/v dilution in blotto for 1 hr, and thendeveloped. The membranes were then stripped with a reducing buffer,blocked in blotto, incubated in anti-Chk2 antibody (Biolegend—San Diego,Calif.) at 1:500 v/v dilution in blotto overnight at 4° C., washed,incubated in HRP conjugated anti-mouse secondary antibody (Jackson) at1:5,000 v/v dilution in blotto for 1 hr, and then developed. Membraneswere developed with PicoWest Developing Solution (Pierce) and exposed tofilm. The intensities of bands were quantified as described in theprevious section. The data are expressed as the average ratio ofpThr68-Chk2 to total Chk2 as normalized to the mitoxantrone control ineach of three independent experiments.

Transwell Invasion Assay: HeLa cells were plated at 250,000 per 35 mmwell and allowed to attach for 8 hr. The cells were washed with PBS andthen treated for 4 hr with the [GI_(99%)] of each compound (or 1%DMSO—vehicle control) in serum free media to decrease the number ofinvading cells in the beginning of the experiment due to a preliminarylack of drug exposure. The cells were then trypsinized and counted bytrypan blue exclusion with a hemacytometer (Sigma—St. Louis, Mo.). Eightmicron pore size control well inserts for 12 well plates (BDBiosciences) were coated with 100 μL 1 mg/mL matrigel (Sigma—St. Louis,Mo.) and allowed to solidify at room temperature for 1 hr. The excessmatrigel was aspirated and the inserts were gently washed with PBS. Thecells were seeded at a density of 300,000 per insert well in a volume of500 μL serum free media containing a derivative or vehicle. The insertswere then placed in wells containing 700 μL serum free media with therespective compound. After 20 hr the cells on the top of the membraneswere gently removed with a cotton swab, the membranes then were fixed inmethanol for 1 min, stained in Gills No.3 Hematoxylin (Sigma—St. Louis,Mo.) for 5 min, rinsed in tap water, and allowed to dry overnight. Themembranes were excised, mounted on slides, and the cells were countedunder the 40× objective of a bright field microscope. The data areexpressed as the average total number of invading cells counted in 20fields of each membrane, as standardized to the vehicle control, fromsix experiments.

Perinucleolar Compartment (PNC) Reduction: HeLa cells (PNC prevalence˜85%) were plated 5,000/well in glass bottom 96 well plates and allowedto attach for 8 hr. The cells were then treated with compounds at therespective [GI_(50%)] for 72 hr and [GI_(99%)] for 20 hr for eachcompound (total media volume was 200 μL/well with 1% DMSO). Aftertreatment cells were immunofluorescently stained as previously described[28], but with a Texas Red labeled secondary antibody (Jackson) insteadof a FITC labeled secondary antibody. PNC prevalence (% ofnon-apoptotic/non-mitotic cells with 1 or more PNCs) was determined byscoring>200 cells per well with fluorescent microscope (60× objective)and images were acquired with the digital camera and image acquisitionsoftware previously mentioned. Scale bar=10 μm. Data are expressed asthe average PNC prevalence (% control) of three individual experiments.

Results and Discussion

Chemical synthesis of derivatives: In experiments conducted during thecourse of development of embodiments for the present invention,derivatives of amonafide that cannot be metabolized by NAT2 (FIG. 1A)while retaining biological activity to potentially dose patients with anamonafide derivative independent of NAT2 genotype or phenotype weredeveloped. Amonafide derivatives with free or protected amines at the6-position, instead of the 5-position of amonafide, were created. Aminoderivatives at the 6-position were synthesized for several reasonsincluding, but not limited to, 1) the chemical synthesis of mostderivatives (A2-A8) had not been described, 2) they are chemically verysimilar to amonafide compared to previously reported derivatives such asmitonafide, azonafide, and elinafide, which do not have aryl amines, and3) the anti-cancer properties of these molecules have not beendescribed. The derivatives synthesized were chosen because alkylation ofthe aryl amine will prevent NAT2 acetylation and also to explore theeffects of hydrophobicity and primary vs. secondary vs. tertiary arylamines on the biological activity of this class of compounds. Aderivative completely lacking the aryl amine (A9) was also synthesizedas a control to determine the biological significance of the aryl amine.The synthesis of A1-A9 was relatively simple and rapid. All reactionswere 1 or 2 steps from commercial reactants to final product. Thehydrogenation of the 6-nitro-imide precursor to produce A1 was the mostefficient reaction at 99%, while the yield for A2-A9 is between 16%-91%(FIG. 1B). This ease of synthesis will allow for rapid synthesis offollow up derivatives in the future if needed.

NAT2 metabolism of derivatives: To determine if the novel derivativesare substrates for NAT2, a NAT2 assay was performed that has beenpreviously described [22]. NAT2*4, a rapid acetylating wild typerecombinant protein [22], was used in solution with its coenzyme,acetylCoA, to determine if it can acetylate amonafide (positivecontrol), A1 (derivative with a free amine), and A2 (representativenegative control with a chemically substituted, or “blocked,” arylamine). The reaction mixtures were incubated for 10 minutes, stopped,solid phase extracted, and then injected into a mass spectrometer todetermine the extent of N-acetylation. Amonafide underwent extensiveacetylation while A2, which lacks the free amine necessary for NAT2activity, was not acetylated (FIG. 1C). A1 was very minimally acetylatedcompared to amonafide, demonstrating that simply moving the aryl amineto the 6-position successfully blocks the acetylation of this chemotype.This finding could be explained, for example, by the aryl amine at the6-position being less sterically favorable for acetylation in theenzymatic pocket, or by the free amine at the 6-position being lessnucleophilic than an amine at the 5-position, or due to a combination ofboth factors. In some embodiments, as the compounds of the presentinvention have chemical similarity with amonafide and an inability to beacetylated by NAT2, the present invention contemplates that thesederivatives may be developed as potential replacements for amonafide oras novel anti-cancer drugs that are not metabolized by NAT2.

Growth inhibition and cancer cell selectivity: Derivatives to beconsidered as potential replacements for amonafide may have, forexample, similar or better anti-cancer activities than amonafide.Therefore the in vitro activities of A1-A9 compared to amonafide byconstructing growth inhibition curves for three human cancer cell lines(HeLa, PC-3M, and MDA-MB-231) was first characterized. The potencies ofthe derivatives were not greatly affected by the chemical alterations atthe 6-position (FIG. 2). A9 still remains fairly potent even though itcompletely lacks an aryl amine, which demonstrates, for example, thearyl amine is not a necessary molecular feature for growth inhibition incell lines; however, the derivatives with a primary or secondary amineare the most potent. These data, along with the known metabolicinactivation of amonafide in humans [13] (FIG. 1A) and previouslyreported in vivo potencies of 5-amino derivatives [17], support that,for example, the dimethylamino group of the molecule is the most activepharmacophore for growth inhibitory activities. However, the chemicalcomposition of the amine in the 6-position can affect the selectivity ofthese compounds. Normal cells (PBMC, HUVEC, and WI-38) were treated withA1-A9 and amonafide to construct GI curves so the cancer cell selectivegrowth inhibition of the derivatives could be compared to amonafide(FIG. 2). PBMCs were chosen as normal cells since the major toxicitiesassociated with amonafide in clinical trials were hematological [4-6, 9]and the other cell lines were chosen since they are non-transformednormal primary endothelial and fibroblastic human cells. Amonafide andmost derivatives are slightly more potent (but not with statisticalsignificance) or equally potent at inhibiting the growth of cancer cellsover normal cells. However, the most hydrophobic derivatives (A5-A7) arepreferentially inhibiting the growth of normal cells with A5 doing sosignificantly (p<0.05), which suggests, for example, that thesecompounds would not be suitable for further development as anti-cancerdrugs (FIG. 2B). In summary, the in vitro potencies of2-[2-(dimethylamino)ethyl]1H-Benz[de]isoquinoline-1,3(2H)-diones are notgreatly affected by the presence of, or chemical alterations of, a6-position aryl amine; however, the selectivity can be altered by thepresence and composition of the amino group in the 6-position.

Subcellular localization of derivatives: Previous studies have showndifferential, side-chain dependent, subcellular localization withazonafide derivatives [18, 26], so it was determined if chemicalalterations at the aryl amine may change subcellular localization of the6-amino derivatives, which can potentially affect the biological actionsof these compounds. The fluorescence of these compounds can be detectedin the green channel (490-520 nm emission filter) of a fluorescentmicroscope, with exception of A9, which does not fluoresce. Therefore,the subcellular localization of these compounds in treated and fixedHeLa cells by epifluorescence microscopy was able to be determined. Thenucleus and cytoplasm were stained to show the relative distribution ofthe compounds within cells (FIG. 3A). All of the compounds localize tothe nucleus to some extent, but the hydrophobic derivatives (A5-A8)appear to localize predominantly in cytoplasmic puncta (FIG. 3). Themore hydrophilic compounds (amonafide and A1-A4) localize predominantlyto the nucleus, with amonafide, A3, and A4 preferentially localizing tothe nucleolus over the nucleoplasm. Subcellular localization patterns donot seem to correlate with the selectivity or potency observed for thesecompounds. Based on the intensity of the fluorescence observed byepifluorescent microscopy and based on the yellow hue (color ofamonafide and A1-A8 in solution) of cells observed by bright fieldmicroscopy, cells treated with the hydrophobic derivatives (A5-A8)attained much higher cellular concentrations at equitoxic doses. Thelarge cellular build up and poor selectivity of the hydrophobicderivatives (A5-A8) indicate that they may not be as efficacious in vivoas they would likely require large doses and hence may cause many sideeffects prior to exerting anti-tumor effects.

DNA intercalation: Amonafide and its structural analogs have been shownto cause topoisomerase II (topo II) dependent DNA damage in purifiedsystems [1, 3].

This activity depends on the ability of these compounds to intercalateDNA, which is the proposed mechanism of cytotoxic action of amonafideand its analogs [1, 3]. Therefore, the intercalation of plasmid DNA byA1-A9 was compared with the intercalation by amonafide to determine ifthey behave similarly. A topoisomerase I (topo I) based plasmid DNAunwinding assay was used to determine the intercalation of each compound[1]. If a DNA intercalating agent is added to this reaction, topo Icannot relax the DNA since the DNA writhe will be altered, leading to anequilibrium that favors the form I (supercoiled) DNA. The results ofthis assay indicated that all derivatives (A1-A9) indeed intercalateDNA; however, A5 intercalates much less than the other compounds,followed by the other hydrophobic derivatives (A6-A8) and A1 (FIG. 4).When topo I is absent from the reaction the DNA band is slightly shiftedup on the gel, further supporting that these compounds are intercalatingDNA.

Topoisomerase II inhibition: Amonafide, mitonafide, and azonafide havebeen all been shown to cause protein linked DNA breaks in vitro [1, 18],which is considered an indicator of topoisomerase inhibition. However,the mechanism by which these compounds inhibit topo-II is not clear. Thecytotoxic mechanism of action of most clinically used topo II inhibitorsis stabilization of topo II-DNA cleavable complexes. These complexes canbe detected in treated cells after genomic DNA extraction [24] anddetection of such cleavable complexes more directly indicates topo IIinhibition than detecting general DNA-protein links. Therefore, an invitro topo II inhibition assay was utilized, which detects such topoII-DNA cleavable complexes (FIG. 5A) in order to determine if amonafideor A1-A9 inhibit topo II by stabilizing the cleavable complex. In thisassay cells are treated with compounds for 20 hours and then lysed toobtain the genomic DNA, and finally the DNA is blotted with topo IIantibodies to determine if any topo II complexes are associated with theDNA. The positive control compounds, mitoxantrone and etoposide, causedformation of abundant drug stabilized topo II-DNA cleavable complexes inHeLa cells (FIG. 5A). Camptothecin (a selective topo I inhibitor) was anegative control and did not cause the stabilization of topo II-DNAcleavable complexes (FIG. 5A). The results show that amonafide and A1-A9treated cells produced very little to no detectable stabilized topoII-DNA cleavable complexes (FIG. 5A). These results suggest, forexample, that amonafide, and structurally related derivatives, do notinhibit topo II in cells by the same mechanism as other topo IIinhibitors. The results are consistent with the past finding thatamonafide is an ATP-independent inhibitor of topo II, in contrast withother well characterized and clinically used topo II inhibitors, whichare ATP-dependent [25]. These findings suggest, for example, that topoII inhibition by amonafide and it derivatives is likely a consequence ofthe DNA intercalation, but not due to direct inhibition of the topo IIenzyme itself. Perhaps the intercalation by this class of compoundsstabilizes the DNA breaks caused by topo II so they can not bereligated, but does not trap the enzyme on the DNA.

Topoisomerase inhibition by most mechanisms should cause activation ofDNA damage response pathways because of the production of single anddouble strand DNA breaks, as is the case with amonafide [3]. It isreasonable to expect that A1-A9 are causing the same type of DNA damageas amonafide since the dimethylamino-imide is the most activepharmacophore of the amonafide molecule [1] and since this pharmacophoreis consistent among the derivatives. Therefore, measuring the DNA damageresponse at a fixed cytotoxic dose ([GI_(99%)]) will determine theextent that DNA damage contributes to the cytotoxicity of thesecompounds. Phosphorylation of Chk2 at threonine68 (Thr68) is an upstreamDNA damage response signaling event, subsequent to the activation ofATR/ATM kinases, which are direct responders to DNA damage by inhibitingthe cell cycle [27]. Activation of Chk2 was determined by measuring theratio of pThr68-Chk2 to total Chk2 in HeLa cells treated with the[GI_(99%)]. Amonafide, A2, and A9 evoked the strongest DNA damageresponse while the most hydrophobic derivatives (A5-A8) caused the leastDNA damage response compared to amonafide (p<0.1, FIG. 5B). This dataqualitatively correlates with the performance of these compounds in theDNA intercalation assay, which supports the hypothesis that topo IIinhibition by these compounds is a result of DNA intercalation ratherthan direct inhibition of topo II.

The differential DNA damage response among the derivatives suggests, forexample, that these compounds may have off-target mechanisms of actionor differential downstream affects. The cytoplasmic localization of thehydrophobic derivatives (A5-A8) suggests, for example, these compoundslikely have cytotoxic mechanisms of action other than DNA intercalation.Amonafide derivatives, for example, likely bind differential DNAsequences, leading to differential biological impacts. For example, somecompounds may preferentially bind gene rich sequences that are importantfor cell proliferation and thereby alter the transcription of thesegenes, leading to a reduction in cellular proliferation. While otherderivatives may inhibit cell growth mainly through the activation of theDNA damage response pathways, such as Chk2 activation, which inhibit thecell cycle. It is likely, for example, that the derivatives inhibit cellgrowth through differential combinations of these two mechanisms or byother mechanisms.

Effect of derivatives on the malignant phenotype—invasion assay: IfA1-A9 have differential targets, DNA binding specificity, or downstreameffects they may differentially alter the malignant behavior of cells.Also, anti-cancer compounds can be efficacious against tumors in otherways besides inhibiting growth and inducing death of cancer cells; theycan also alter the behavior of malignant cells. Therefore, the abilityof A1-A9 and amonafide to alter the invasive behavior of cancer cells ina transwell (also known as Boyden chamber) invasion assay was evaluated,which determines the ability of cells to migrate through a proteinaciousgel. This behavior is considered to be representative of cancer cellinvasion through the basement membrane of the primary tumor environment.In this assay, treated cells are seeded on to the top of thematrigel-coated porous membrane, allowed to invade through theproteinacious gel onto the underside of the membrane, and then counted.Amonafide, A1, A3, and A8 did not significantly alter the invasivebehavior of HeLa cells compared to the control, while the otherderivatives significantly (p<0.05) decreased the invasiveness of HeLacells to varying extents (FIG. 6A). The anti-invasive properties ofthese compounds show no obvious correlations between any of the otherdata presented here, which demonstrates, for example, that thesecompounds are differentially affecting cellular behavior and furthersuggests, for example, these derivatives have differential off target ordownstream effects.

Effect of derivatives on the malignant phenotype—PNC prevalencereduction: It was discovered that amonafide was able to eliminate aphenotypic marker of metastatic cells, called the perinucleolarcompartment (PNC). The PNC is a unique structural marker that it is onlyfound in malignantly transformed cancer cells, irrespective of tissueorigin, and indicates cells that are capable of metastasis [28-30]. Thissuggests, for example, that the PNC is a more comprehensive marker ofmalignancy than currently used molecular markers of malignancy, whichare often cancer type specific and not directly involved in promotingthe malignant behavior. Previous studies indicate that the PNC likelyconveys a functional metastatic advantage to cancer cells [28-30]. ThePNC prevalence (% of cells with one or more PNC) in human breast cancerpositively correlates with the progression of the disease and is alwaysnear 100% in distant metastases [30]. This suggests, for example, thatPNC elimination may contribute to activity of amonafide againstadvanced/metastatic breast cancers in clinical trials [4-6]. Therefore,PNC prevalence reduction of amonafide and A1-A9 for was quantified tworeasons: 1) to determine which derivatives will likely be effectiveagainst advanced/metastatic cancers and 2) to determine if there isdifferential PNC reducing activity among the derivatives and, if so,determine if it correlates with other biological activities of thesecompounds. The PNC prevalence reduction was determined for amonafide andA1-A9 at the respective [GI_(50%)] and [GI_(99%)] doses in HeLa cells,which have a high PNC prevalence of ˜85%. At the [GI_(99%)] amonafideand all the derivatives, with the exception of A5, were able to greatlyreduce the PNC prevalence (FIG. 6B). Amonafide, A1, and A4 were the onlycompounds that were able effectively reduce the PNC at their [GI_(50%)]doses, which suggests, for example, that A1 and A4 are the most likelyto share similar anti-cancer properties of amonafide and potentiallyhave the best efficacy against metastatic cancers. Again, it was foundthat these derivatives cause differential biological responses at fixedcytotoxic doses, which supports differential targets or downstreamaffects of these compounds. Also, PNC prevalence reduction does notcorrelate with DNA intercalation, DNA damage response, or growthinhibition, which demonstrates that PNC elimination by these derivativesis due to specific molecular interactions of the compounds with cellulartargets, not simply a side effect of growth inhibition.

A4 is a strong candidate for further development as it has similarpotency and selectivity as amonafide, shows near identical sub-cellularlocalization to amonafide, reduces PNC prevalence to the same extent asamonafide at the [GI_(50%)], and greatly decreases invasion compared toamonafide and control. The PNC prevalence reduction and inhibition ofinvasive behavior by A4 suggests, for example, this compound may beefficacious against metastatic and advanced tumors. A1 also deservesfurther development as its potency, selectivity, sub-cellularlocalization, PNC prevalence reduction, chemical structure, andphysio-chemical properties are near identical to amonafide, whichsuggests, for example, the biodistribution in animals will be verysimilar. Although originally included in these studies as a control todetermine the biological significance of the 5 and 6-position amines, A9also deserves further consideration for further development since itspotency, selectivity, and DNA damage activation are similar to amonafideand since it decreases invasive behavior.

While these 6-amino amonafide derivatives, named here as numonafides,are chemically very similar to amonafide, the small chemical differencesbetween numonafides and amonafide cause differential in vitro biologicalactivities likely due, for example, to differential off-target, DNAbinding specificities, or downstream affects. Also, the chemicaldifferences among the derivatives may cause these compounds to becomesubstrates for metabolic enzymes other than NAT2 and CYP1A2 and maydifferentially affect their biodistribution and clearance in an animal.

INCORPORATION BY REFERENCE The entire disclosure of each of the patentdocuments and scientific articles referred to herein is incorporated byreference for all purposes. Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

REFERENCES

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1. A composition comprising a compound described by the followingformula:

including salts, esters, and prodrugs thereof; and including both R andS enantiomeric forms and racemic mixtures thereof; wherein X is presentor absent, and if present is

wherein R is H, ethyl, or R1, and wherein R1 is H, ethyl,


2. The composition of claim 1, wherein said compound is selected fromthe group consisting of:


3. A pharmaceutical preparation comprising one or more of the compoundsdescribed in claim 1 and a pharmaceutically acceptable carrier.
 4. Amethod of treating a hyperproliferative disorder comprisingadministering an effective amount of the pharmaceutical preparation ofclaim 1 to a subject in need thereof.
 5. The method of claim 4, whereinsaid hyperproliferative disorder is a cancer.
 6. The method of claim 5,wherein said cancer is a tumor, a neoplasm, a lymphoma, or a leukemia.7. The method of claim 6, further comprising co-administering to saidsubject an anti-cancer agent.
 8. The method of claim 7, wherein saidanti-cancer agent is select from the group consisting of Acivicin;Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin;Aldesleukin; Alitretinoin; Allopurinol Sodium; Altretamine; Ambomycin;Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole;Annonaceous Acetogenins; Anthramycin; Asimicin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bexarotene;Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin;Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Bullatacin; Busulfan;Cabergoline; Cactinomycin; Calusterone; Caracemide; Carbetimer;Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin;Cedefingol; Celecoxib; Chlorambucil; Cirolemycin; Cisplatin; Cladribine;Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA(N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin;Daunorubicin Hydrochloride; Daunomycin; Decitabine; Denileukin Diftitox;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized OilI 131; Etoposide; Etoposide Phosphate; Etoprine; FadrozoleHydrochloride; Fazarabine; Fenretinide; Floxuridine; FludarabinePhosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; FostriecinSodium; FK-317; FK-973; FR-66979; FR-900482; Gemcitabine; GeimcitabineHydrochloride; Gemtuzumab Ozogamicin; Gold Au 198; Goserelin Acetate;Guanacone; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-1a; Interferon Gamma-1b; Iproplatin;Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; LeuprolideAcetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine;Losoxantrone Hydrochloride; Masoprocol; Maytansine; MechlorethamineHydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan;Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Methoxsalen; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin;Mitogillin; Mitomalcin; Mitomycin; Mytomycin C; Mitosper; Mitotane;Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;Oprelvekin; Ormaplatin; Oxisuran; Paclitaxel; Pamidronate Disodium;Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rituximab; Rogletimide; Rolliniastatin;Safingol; Safingol Hydrochloride; Samarium/Lexidronam; Semustine;Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Squamocin; Squamotacin;Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur;Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine;Tomudex; TOP-53; Topotecan Hydrochloride; Toremifene Citrate;Trastuzumab; Trestolone Acetate; Triciribine Phosphate; Trimetrexate;Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; UracilMustard; Uredepa; Valrubicin; Vapreotide; Verteporfin; Vinblastine;Vinblastine Sulfate; Vincristine; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride; 2-Chlorodeoxyadenosine; 2′-Deoxyformycin;9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid;2-chloro-2′-arabino-fluoro-2′-deoxyadenosine;2-chloro-2′-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R;CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlorethamine);cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan;N-methyl-N-nitrosourea (MNU); N,N′-Bis(2-chloroethyl)-N-nitrosourea(BCNU); N-(2-chloroethyl)-N′-cyclohex-yl-N-nitrosourea (CCNU);N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU);N-(2-chloroethyl)-N′-(diethyl)ethylphosphonate-N-nit-rosourea(fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide;temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; Cisplatin;Carboplatin; Ormaplatin; Oxaliplatin; C1-973; DWA 2114R; JM216; JM335;Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine;6-Mercaptopurine; 6-Thioguanine; Hypoxanthine; teniposide; 9-aminocamptothecin; Topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin;darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D);amsacrine; pyrazoloacridine; all-trans retinol;14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-Hydroxyphenyl)retinamide; 13-cis retinoic acid; 3-Methyl TTNEB; 9-cis retinoic acid;fludarabine (2-F-ara-AMP); 2-chlorodeoxyadenosine (2-Cda),Antiproliferative agents, Piritrexim Isothionate, Antiprostatichypertrophy agents, Sitogluside, Benign prostatic hyperplasia therapyagents, Tamsulosin Hydrochloride, Prostate growth inhibitor agents,Pentomone, and Radioactive agents, Fibrinogen 1 125; Fludeoxyglucose F18; Fluorodopa F 18; Insulin I 125; Insulin I 131; Iobenguane I 123;Iodipamide Sodium I 131; Iodoantipyrine I 131; Iodocholesterol I 131;Iodohippurate Sodium I 123; Iodohippurate Sodium I 125; IodohippurateSodium I 131; Iodopyracet I 125; Iodopyracet I 131; IofetamineHydrochloride I 123; Iomethin I 125; Iomethin I 131; Iothalamate SodiumI 125; Iothalamate Sodium I 131; Iotyrosine I 131; Liothyronine I 125;Liothyronine I 131; Merisoprol Acetate Hg 197; Merisoprol Acetate Hg203; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Tc 99mAntimony Trisulfide Colloid; Technetium Tc 99m Bicisate; Technetium Tc99m Disofenin; Technetium Tc 99m Etidronate; Technetium Tc 99mExametazime; Technetium Tc 99m Furifosmin; Technetium Tc 99m Gluceptate;Technetium Tc 99m Lidofenin; Technetium Tc 99m Mebrofenin; Technetium Tc99m Medronate; Technetium Tc 99m Medronate Disodium; Technetium Tc 99mMertiatide; Technetium Tc 99m Oxidronate; Technetium Tc 99m Pentetate;Technetium Tc 99m Pentetate Calcium Trisodium; Technetium Tc 99mSestamibi; Technetium Tc 99m Siboroxime; Technetium Tc 99m Succimer;Technetium Tc 99m Sulfur Colloid; Technetium Tc 99m Teboroxime;Technetium Tc 99m Tetrofosmin; Technetium Tc 99m Tiatide; Thyroxine I125; Thyroxine I 131; Tolpovidone I 131; Triolein I 125; and Triolein I131.
 9. A pharmaceutical preparation comprising one or more of thecompounds described in claim 2 and a pharmaceutically acceptablecarrier.